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WO1999039743A2 - Procede de stimulation de la proliferation des cellules hepatiques - Google Patents

Procede de stimulation de la proliferation des cellules hepatiques Download PDF

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Publication number
WO1999039743A2
WO1999039743A2 PCT/US1999/002618 US9902618W WO9939743A2 WO 1999039743 A2 WO1999039743 A2 WO 1999039743A2 US 9902618 W US9902618 W US 9902618W WO 9939743 A2 WO9939743 A2 WO 9939743A2
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seq
group
tyr
active agent
ala
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PCT/US1999/002618
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WO1999039743A3 (fr
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Kathleen E. Rodgers
Gere Dizerega
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University Of Southern California
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Priority to AU25903/99A priority Critical patent/AU2590399A/en
Publication of WO1999039743A2 publication Critical patent/WO1999039743A2/fr
Publication of WO1999039743A3 publication Critical patent/WO1999039743A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/085—Angiotensins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/32—Angiotensins [AT], angiotensinogen

Definitions

  • This present invention relates to methods, cell culture media, and kits for use in accelerating the proliferation of hepatic cells.
  • liver is an epithelial organ that contains two major differentiated cell types: the hepatocyte and the bile ductule cell, which are thought to arise from a common hepatic stem cell.
  • hepatocyte and the bile ductule cell, which are thought to arise from a common hepatic stem cell.
  • Hepatic cells are typically quiescent in adults.
  • liver tissue to regenerate has been known for some time and is controlled by cytokines of paracrine and/or autocrine origin which either stimulate or inhibit hepatic cell growth.
  • Hepatic stem cells whose immediate progeny are referred to as oval cells or small epithelial cells (hereinafter referred to as 'SEC'), assume the burden of regenerative liver growth only after significant hepatocyte loss.
  • liver failure In contrast to liver regeneration after PH, hepatic regeneration in acute liver failure (also known as fulminant hepatic failure) occurs by the proliferation and differentiation of hepatic stem cells. (Kay and Fausto, Mol. Med. Today 3:108-115 (1997); hereby incorporated by reference in its entirety.) Acute liver failure can be caused by several distinct diseases, including viral hepatitis, chemical injury (for example, caused by acetaminophen/paracetamol), Wilson's disease, and Reye's syndrome (Id.) Oval cells or SEC arise initially in the bile epithelium after activation of hepatic stem cells following physiologically- or experimentally-induced liver damage (Alison et al., 1996; Thorgeirsson, 1996; Kay and Faust, 1997; and references therein).
  • the liver cannot be classified as a classical stem cell-fed tissue, like the epidermis, the intestinal epithelium, the hematopoietic system, or the bone marrow. Unlike other tissues capable of regeneration (e.g., skin and bone marrow), regeneration of the liver is not dependent upon small populations of stem cells or progenitor cells. However, large numbers of hepatic stem cells do appear when mature hepatocytes are inhibited from proliferation (Thorgeirsson, 1996; Sell, Mod. Pathol. 7:105 (1994); Fausto, et al, Proc. Soc. Exp. Biol. Med. 204:237 (1993)). A number of acute and chronic conditions can lead to the damage of liver tissue.
  • hepatocarcinoma hepatitis infection
  • cirrhosis of the liver partial hepatectomy
  • fulminant hepatic failure hepatocyte transplantation
  • liver transplantation the survival of patients with these disorders depends upon successful liver regeneration, which occurs through the proliferation of hepatocytes and/or hepatic stem cells.
  • hepatic stem cells and hepatocytes are provided. Such methods will provide clinical benefits in liver regeneration following resection of hepatocarcinomas, hepatitis infection, cirrhosis of the liver, partial hepatectomy, fulminant hepatic failure, hepatocyte transplantation, liver transplantation, and other hepatic disorders where rapid regeneration of the liver is desirable. Such methods are also useful in rapidly providing a large population of hepatic cells for use in cell therapy and for providing a large population of transfected hepatic cells for use in gene therapy.
  • the present invention provides methods that promote hepatic cell proliferation by contacting the cells with angiotensinogen, angiotensin I (hereinafter referred to as 'Al'), Al analogues, and/or Al fragments and analogues thereof, angiotensin II (hereinafter referred to as 'All') analogues, All fragments or analogues thereof, and/or All AT 2 type 2 receptor agonists, either alone or in combination with other growth factors and cytokines.
  • angiotensinogen hereinafter referred to as 'Al'
  • Al analogues Al analogues
  • angiotensin II hereinafter referred to as 'All'
  • an improved cell culture medium for the proliferation of hepatic cells, wherein the improvement comprises addition to the cell culture medium of an effective amount of angiotensinogen, Al, Al analogues, and/or Al fragments and analogues thereof, All analogues, All fragments or analogues thereof, and/or All AT type 2 receptor agonists.
  • kits for the propagation of hepatic cells comprising an effective amount of angiotensinogen, Al, Al analogues, and/or Al fragments and analogues thereof, All analogues, All fragments or analogues thereof, and or All AT type 2 receptor agonists, and instructions for culturing the cells.
  • kits for the propagation of hepatic cells comprise an effective amount of angiotensinogen, Al, Al analogues, and/or Al fragments and analogues thereof, All analogues, All fragments or analogues thereof, and or All AT type 2 receptor agonists, and instructions for culturing the cells.
  • Preferred embodiments of the kit further comprise cell culture growth medium, a sterile container, and an antibiotic supplement.
  • hepatic cells refers to hepatic stem cells that are poorly differentiated, demonstrate extensive proliferation, and have multiple differentiation options (Thorgeirsson, FASEB J. 10:1249 (1996)), as well as hepatocytes and bile ductule cells derived therefrom.
  • hepatic stem cells include, but are not limited to, SEC cells, oval cells, as well as cultured WB-F344 cells
  • proliferation encompasses both cell self renewal and cellular proliferation with accompanying differentiation.
  • active agents refers to the group of compounds comprising angiotensinogen, angiotensin I (Al), Al analogues, Al fragments and analogues thereof, angiotensin II analogues, All fragments or analogues thereof and All AT 2 type 2 receptor agonists.
  • angiotensin The biological formation of angiotensin is initiated by the action of renin on the plasma substrate angiotensinogen (Circulation Research 60:786-790 (1987); Clouston et al., Genomics 2:240-248 (1988); Kageyama et al, Biochemistry 23:3603-3609; Ohkubo et al., Proc. Natl. Acad. Sci. 80:2196-2200 (1983); all references hereby incorporated in their entirety).
  • the substance so formed is a decapeptide called angiotensin I (Al) which is converted to All by the converting enzyme angiotensinase which removes the C-terminal His-Leu residues from Al, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu [SEQ ID NO:37]. All is a known pressor agent and is commercially available.
  • All may accelerate wound repair through increased neovascularization, growth factor release, reepithelialization and/or production of extracellular matrix.
  • All analogues and fragments, AT2 agonists, as well as AIII and AIII analogues and fragments in wound healing has also been described. (U.S. Patent No. 5,629,292; U.S. Patent No. 5,716,935; WO 96/39164; all references herein incorporated by reference in their entirety.)
  • angiotensinogen, Al, Al analogues, and/or Al fragments and analogues thereof, All analogues, All fragments or analogues thereof, and/or All AT 2 type 2 receptor agonists accelerate the proliferation of hepatic stem cells, hepatocytes, or bile ductule cells.
  • All fragment AII(l-7) acts through a receptor(s) that is distinct from the ATI and AT2 receptors which modulate All activity.
  • a peptide agonist selective for the AT2 receptor (All has 100 times higher affinity for AT2 than ATI) is p-aminophenylalanine6-AII ["(p-NH 2 -Phe)6-AII)"], Asp- Arg-Val-Tyr-Ile-Xaa-Pro-Phe [SEQ ID NO.36] wherein Xaa is p-NH 2 -Phe (Speth and Kim, BBRC 169:997-1006 (1990).
  • This peptide gave binding characteristics comparable to AT2 antagonists in the experimental models tested (Catalioto, et al., Eur. J. Pharmacol. 256:93-97 (1994); Bryson, et al, Eur. J. Pharmacol. 225:119-127 (1992).
  • AII(l-7) All residues 1-7) or other fragments of All to evaluate their activity.
  • AII(l-7) elicits some, but not the full range of effects elicited by All (Pfeilschifter, et al., Eur. J. Pharmacol. 225:57-62 (1992); Jaiswal, et al., Hypertension 19 (Supp. II):II-49-II-55 (1992); Edwards and Stack, J. Pharmacol. Exper. Ther. 266:506-510 (1993); Jaiswal, et al., J. Pharmacol. Exper. Ther.
  • a preferred class of AT2 agonists for use in accordance with the present invention comprises Al, All, and Al or All analogues or active fragments thereof having p-NH-Phe in a position corresponding to a position 6 of AIL
  • various nonpeptidic agents e.g., peptidomimetics
  • having the requisite AT2 agonist activity are further contemplated for use in accordance with the present invention.
  • the active All analogues, fragments of All and analogues thereof of particular interest in accordance with the present invention are characterized as comprising a sequence consisting of at least three contiguous amino acids of groups R -R in the sequence of general formula I
  • R A is suitably selected from Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala, Me 2 Gly, Pro, Bet, Glu(NH 2 ), Gly, As ⁇ (NH 2 ) and Sue,
  • R B is suitably selected from Arg, Lys, Ala, Orn, Ser(Ac), Sar, D-Arg and D-Lys
  • R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr;
  • R 4 is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;
  • R 5 is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly;
  • R 6 is His, Arg or 6-NH 2 -Phe;
  • R 7 is Pro or Ala;
  • R 8 is selected from the group consisting of Phe, Phe(Br), He and Tyr, excluding sequences including R 4 as a terminal Tyr group, wherein the active agent is not AIL
  • Compounds falling within the category of AT2 agonists useful in the practice of the invention include the AH analogues set forth above subject to the restriction that R is p-NH 2 -Phe.
  • Particularly preferred combinations for R A and R B are Asp- Arg, Asp-Lys, Glu-
  • AIII Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:2]; AH(3-8), also known as desl-AIII or AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:3]; AII(l-7), Asp-Arg-Val- Tyr-Ile-His-Pro ⁇ SEQ ID NO:4]; AII(2-7).
  • Arg-norLeu- Tyr-Ile-His-Pro-Phe [SEQ ID NO: 12] and Arg-Val-Tyr-norLeu-His-Pro-Phe [SEQ ID NO: 13].
  • Still another preferred embodiment encompassed within the scope of the invention is a peptide having the sequence Asp-Arg-Pro-Tyr-Ile-His-Pro-Phe [SEQ ID NO:31].
  • AII(6-8), His-Pro-Phe [SEQ ID NO:14] and AII(4-8), Tyr-Ile-His-Pro-Phe [SEQ ID NO: 15] were also tested and found not to be effective.
  • a class of particularly preferred compounds in accordance with the present invention consists of those with the following general structure:
  • Rl-Arg-R2-R3-R4-His-Pro-R5 wherein Rl is selected from the group consisting of H and Asp;
  • R2 is selected from the group consisting of Val and Pro;
  • R3 is selected from the group consisting of Tyr and Tyr(PO 3 ) 2 ;
  • R4 is selected from the group consisting of Ala, He, Leu, and norLeu;
  • R5 is Phe, He, or is absent, and wherein the active agent is not AIL
  • Particularly preferred embodiment of this class are selected from the group consisting of SEQ ID NO:4, SEQ ID NO: 18, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID NO:38
  • R 2 is selected from the group consisting of H, Arg, Lys, Ala,
  • R 3 is selected from the group consisting of Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr;
  • R 4 is selected from the group consisting of Tyr, Tyr(PO 3 ) 2 , Thr, Ser, homoSer and azaTyr;
  • R 5 is selected from the group consisting of He, Ala, Leu, norLeu, Val and Gly;
  • R 6 is His, Arg or 6-NH 2 -Phe;
  • R 7 is Pro or Ala
  • R 8 is selected from the group consisting of Phe, Phe(Br), He and Tyr.
  • a particularly preferred subclass of the compounds of general formula II has the formula
  • R 2 , R 3 and R 5 are as previously defined.
  • Particularly preferred is a compound of the formula Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO:38].
  • Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-Pro- Phe [SEQ ID NO: 17] and Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID NO:18].
  • R 2 Appropriate side chains on the amino acid in position R 2 may contribute to affinity of the compounds for target receptors and/or play an important role in the conformation of the peptide. For this reason, Arg and Lys are particularly preferred as R 2 .
  • R 3 may be involved in the formation of linear or nonlinear hydrogen bonds with R 5 (in the gamma turn model) or R 6 (in the beta turn model). R 3 would also participate in the first turn in a beta antiparallel structure (which has also been proposed as a possible structure). In contrast to other positions in general formula I, it appears that beta and gamma branching are equally effective in this position. Moreover, a single hydrogen bond may be sufficient to maintain a relatively stable conformation. Accordingly, R may suitably be selected from Val, Ala, Leu, norLeu, He, Gly, Pro, Aib, Acpc and Tyr.
  • R 4 is preferably selected from Tyr, Thr, Tyr (PO 3 ) 2 , homoSer, Ser and azaTyr.
  • Tyr is particularly preferred as it may form a hydrogen bond with the receptor site capable of accepting a hydrogen from the phenolic hydroxyl (Regoli, et al. (1974), supra).
  • an amino acid with a ⁇ aliphatic or alicyclic chain is particularly preferred
  • Gly is suitable in position R 5 , it is preferred that the amino acid in this position be selected from He, Ala, Leu, norLeu, Gly and Val.
  • R 6 is His, Arg or 6-NH 2 -Phe.
  • the unique properties of the imidazole ring of histidine e.g., ionization at physiological pH, ability to act as proton donor or acceptor, aromatic character) are believed to contribute to its particular utility as R 6 .
  • conformational models suggest that His may participate in hydrogen bond formation (in the beta model) or in the second turn of the antiparallel structure by influencing the orientation of R 7 .
  • R 7 should be Pro in order to provide the most desirable orientation of
  • Analogues of particular interest include the following: TABLE 2: Angiotensin II Analogues
  • polypeptides of the instant invention may be synthesized by methods such as those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, 111. (1984) and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) for solution synthesis.
  • the disclosures of the foregoing treatises are incorporated by reference herein.
  • these methods involve the sequential addition of protected amino acids to a growing peptide chain (U.S. Patent No. 5,693,616, herein incorporated by reference in its entirety). Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage. After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.
  • a method of increasing in vitro and ex vivo hepatic cell proliferation by exposure to angiotensinogen, Al, Al analogues, and/or Al fragments and analogues thereof, AH analogues, AH fragments or analogues thereof, and/or All AT type 2 receptor agonists ("active agents") is disclosed.
  • Experimental conditions for the isolation, purification, ex vivo growth and in vivo mobilization of hepatic stem cells and hepatocytes have been reported (Drakes et al., J. Immunol. 159:4268 (1997); Omori et al. Hepatology 26:720, (1997); U.S. Patent No. 4,914,032; U.S. Patent No. 5,227,158; all references incorporated by reference herein in their entirety) .
  • hepatocytes are isolated using procedures known in the art. (for example, see Free Radical Biology and Medicine 18:303-310). The isolated hepatocytes are resuspended in appropriate tissue culture medium to induce cell adherence. After 5-7 days in culture, the cells are treated with the active agents at various concentrations, preferably ranging from 0.1 ng/ml to 10 mg/ml. Cell proliferation is assessed at various time points during culture using methods well known in the art, including, but not limited to, measuring the rate of DNA synthesis according to the method of Nakamura and coworkers (Nakamura et al., J. Biochem. (Tokyo) 94:1029 (1982); Nakamura et al., Biochem.
  • hepatic stem cells are isolated from non-transplantable adult mammalian livers, particularly from the bile duct epithelium (Drakes et al, J. Immunol. 159:4268 (1997); Omori et al. Hepatology 26:720, (1997)). Hepatic stem cells are then selected for in these samples and cultured under appropriate growth conditions, in the presence of the active agents of the invention. Hepatic stem cell proliferation is assessed at various time points during culture as described above.
  • hepatic stem cells are isolated from adult mammalian livers by centrifugal elutriation of dispersed liver tissue (Hayner et al., Cancer Res. 44:332 (1984); Germain et al., Cancer Res. 45:673 (1985) Germain et al., Cancer Res. 48:368 (1988); Drakes et al., J. Immunol. 159:4268 (1997); Omori et al., Hepatology 26:720 (1997)).
  • adult mammalian liver is perfused in situ via the portal vein in two steps, first with Mg + - and Ca 2+ -free Hank's balanced salt solution immediately followed by 0.1% pronase and 0.1% collagenase in buffered William's solution.
  • Dispersed liver cells are then successively filtered on 240mm-pore and 70mm-pore nylon screens.
  • the parenchymal cell fraction is obtained after multiple washings by centrifugation at 50 x g for 3 minutes.
  • the nonparenchymal cell fraction is obtained by centrifugation at 200 x g for 10 minutes of the supernatant from which hepatocytes had been repeatedly eliminated by centrifugation.
  • hepatic stem cells are suspended in standard culture medium, including but not limited to, RPMI-1640 (Gibco BRL, MD) and incubated in the presence of, preferably, between about 0.1 ng/ml and about 10 mg/ml of the active agents of the invention.
  • the cells are expanded for a period of between 8 and 21 days, then cellular proliferation is monitored by measuring the rate of DNA synthesis (Nakamura et al., 1982; Nakamura et al., 1984).
  • Differentiation of hepatic stem cells to hepatocytes or bile ductule cells is detected by measuring the expression of typical differentiated hepatocyte markers including, but not limited to, albumin, transferrin, and alpha- 1-antitrypsin, using standard methods well known in the art. These methods can include, but are not limited to, Northern blot analyses or reverse transcriptase-polymerase chain reaction (RT-PCR) with marker-specific DNA primers (Omori et al., Hepatology 25:1115 (1997); Drakes et al., J. Immunol. 159:4268 (1997); Omori et al., Hepatology 26:720 (1997)). Similarly, "self-renewal" of hepatic stem cells is assessed periodically by phase-contrast light microscopy and or immunohistochemistry (Overturf et al., Am. J. Pathol. 151 :5 (1997)).
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • hepatic stem cells, bile ductule cells, or hepatocytes that have been cultured in the presence of the active agents are used for ex vivo liver-directed gene therapy (Alt and Cushman, J. Hepatol. 23:746 (1995)).
  • the cells Prior to ex vivo liver-directed gene therapy, the cells are rinsed to remove all traces of culture fluid, resuspended in an appropriate medium and then pelleted and rinsed several times. After the final rinse, the cells are resuspended at between 0.7 x 10 6 and 50 x 10 6 cells per ml in an appropriate medium and reinfused into a subject through intravenous infusions. Following reinfusion, hepatic cell proliferation is assessed by the methods discussed above.
  • assessment of the in vivo proliferative effect of the active agents of the invention on hepatic cells is performed by histochemical evaluations of the liver including, but not limited to, hepatocytic parenchyma, periportal parenchyma, biliary epithelium, hyperplastic ducts within the periportal zone, arborizing ductules, and the portal spaces.
  • in vivo proliferation of hepatic cells is assessed by reactivity to an antibody directed against a protein known to be present in higher concentrations in proliferating cells than in non-proliferating cells, such as proliferating cell nuclear antigen (PCNA or cyclin; Zymed Laboratories) (Rodgers et al., J. Burn Care Rehabil. 18:381-388 (1997); incorporated herein by reference in its entirety).
  • PCNA proliferating cell nuclear antigen
  • the active agents are used to increase in vivo hepatic cell proliferation.
  • the active agents may be administered by any suitable route, including orally, parenterally, by inhalation spray, rectally, transdermally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • parenteral as used herein includes, subcutaneous, intravenous, intraarterial, intramuscular, intrastemal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or intraperitoneally.
  • the active agents may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions) and may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc.
  • active agents of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other compounds.
  • the active agents and other compounds can be formulated as separate compositions that are given at the same time or different times, or the active agents and other compounds can be given as a single composition.
  • the active agents are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration.
  • the active agents may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and or polyvinyl alcohol, and tableted or encapsulated for conventional administration.
  • the active agents of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.
  • Other adjuvants and modes of administration are well known in the pharmaceutical art.
  • the carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions,
  • ointments creams, or pastes
  • drops suitable for administration to the eye, ear, or nose.
  • the dosage regimen for increasing in vivo proliferation of hepatic cells with the active agents of the invention is based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed.
  • the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Dosage levels of the order of between 0.1 ng/kg and 10 mg/kg of the active agents per body weight are useful for all methods of use disclosed herein.
  • the treatment regime will vary depending on the condition being treated, based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration, and the particular compound employed.
  • the active agents are administered parenterally.
  • a suitable parenteral dose of the active agents is preferably between about 0.1 ng/kg and about 10 mg/kg administered twice daily.
  • the active agent may comprise from 0.001% to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation.
  • an improved cell culture medium is provided for the proliferation of hepatic cells, wherein the improvement comprises addition to the cell culture medium of an effective amount of the active agents of the invention.
  • Any cell culture media that can support the growth of hepatic cells can be used with the present invention.
  • Such cell culture media include, but are not limited to Basal Media Eagle, Dulbecco's Modified Eagle Medium, Iscove's Modified Dulbecco's Medium, McCoy's Medium, Minimum Essential Medium, F-10 Nutrient Mixtures, Opti-MEM® Reduced-Serum Medium, and RPMI Medium, or combinations thereof.
  • the improved cell culture medium can be supplied in either a concentrated (ie: 10X) or non-concentrated form, and may be supplied as a liquid, a powder, or a lyophilizate.
  • the cell culture may be either chemically defined, or may contain a serum supplement.
  • Culture media and serum supplements are commercially available from many sources, such as GIBCO BRL (Gaithersburg, MD) and Sigma (St. Louis, MO)
  • kits for the propagation of hepatic cells wherein the kits an effective amount of the active agents of the invention, and a set of instructions for culturing the cells.
  • the kit further comprises cell culture media.
  • Any cell culture media that can support the growth of hepatic cells can be used with the present invention. Examples of such cell culture media are described above.
  • the improved cell culture medium can be supplied in either a concentrated (ie:
  • the cell culture may be either chemically defined, or may contain a serum supplement.
  • the kit of the present invention further comprises a sterile container.
  • the sterile container can comprise either a sealed container, such as a cell culture flask, a roller bottle, or a centrifuge tube, or a non- sealed container, such as a cell culture plate or microtiter plate (Nunc; Naperville, IL).
  • the kit further comprises an antibiotic supplement for inclusion in the reconstituted cell growth medium.
  • antibiotic supplements include, but are not limited to actimonycin D,
  • Fungizone® Fungizone®, kanamycin, neomycin, nystatin, penicillin, streptomycin, or combinations thereof (GIBCO).
  • liver was minced and placed in culture with 0.02% Type VIII collagenase (Sigma, St. Louis, MO) in RPM 1640 overnight at 37° C. Thereafter, the cells were collected in RPMI 1640 medium containing 1 mg/ml bovine serum albumin and 5 ⁇ g/ml bovine insulin. The cell suspension was filtered through sterile gauze and allowed to sediment for 20 minutes to remove cellular debris and blood. The cells were then washed three times by centrifugation at 1200 rpm. Viability was assessed by Trypan blue exclusion.
  • the hepatocytes were resuspended in 75%) Eagle's minimal essential medium/25%) Medium 199 (Gibco BRL) buffered with sodium bicarbonate and supplemented with 10%> fetal calf serum containing 50 ⁇ g/ml streptomycin, 7.5 IU/ml penicillin, 5 ⁇ g/ml insulin, and 1 mg/ml bovine serum albumin at 1 x 10 6 cells/ml. The medium was changed after 3-4 hours of adherence and was changed again after 24 hours. The cells were cultured for 5-7 days prior to use in the following studies.
  • the cells were detached from the tissue culture plastic with 0.05%) trypsin-EDTA (Gibco-BRL Products) at 37°C for 10 minutes.
  • the cells were washed one time with sterile phosphate buffered saline (pH 7.2) and adjusted to a concentration of 200 cells/ml. Two hundred ⁇ l of this suspension was aliquoted into wells of 96 well plates and allowed to attach to the tissue culture plastic. After adherence, AH, All analogues and AH fragments were added to the wells in duplicate to a final concentration of 10 ⁇ g/ml.
  • the identity of the AH analogues and fragments is shown in Table 1.
  • the present invention by providing methods for enhanced proliferation of hepatic cells, will greatly increase the clinical uses of hepatic cells for liver regeneration after resection of hepatocarcinomas and in other hepatic disorders where a more rapid regeneration of the liver is desirable. This is true both for increased “self- renewal", which will provide a larger supply of hepatic stem cells capable of generating additional hepatocytes, epithelial cells, and/or exocrine pancreatic cells and for proliferation with differentiation, which will provide a larger supply of epithelial cells, hepatocytes, or exocrine pancreatic cells, for use in liver regeneration.
  • the method of the present invention also increases the potential utility of hepatic cells as vehicles for gene therapy in hepatic system disorders by more efficiently providing a large number of such cells for transfection, and also by providing a more efficient means to rapidly expand transfected hepatic cells.

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Abstract

La présente invention concerne un milieu de culture cellulaire amélioré ainsi que des procédés et des kits destinés à stimuler la prolifération et la différentiation des cellules hépatiques par une croissance en présence de l'angiotensinogène, l'angiotensine I (AI), des analogues de AI, et/ou des fragments de AI et des analogues de ceux-ci, des analogues de l'angiotensine II (AII), des fragments de AII ou des analogues de ceux-ci, et/ou des agonistes du récepteur de AII du type 2 (AT2), seuls ou en association avec d'autres facteurs de croissance et de cytokines.
PCT/US1999/002618 1998-02-09 1999-02-08 Procede de stimulation de la proliferation des cellules hepatiques WO1999039743A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU25903/99A AU2590399A (en) 1998-02-09 1999-02-08 Method of promoting hepatic cell proliferation

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US7410498P 1998-02-09 1998-02-09
US60/074,104 1998-02-09
US10841298P 1998-11-13 1998-11-13
US60/108,412 1998-11-13

Publications (2)

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WO1999039743A2 true WO1999039743A2 (fr) 1999-08-12
WO1999039743A3 WO1999039743A3 (fr) 1999-09-30

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PCT/US1999/002618 WO1999039743A2 (fr) 1998-02-09 1999-02-08 Procede de stimulation de la proliferation des cellules hepatiques

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AU (1) AU2590399A (fr)
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050048A3 (fr) * 1999-02-26 2001-02-01 Univ Pittsburgh Transplantation de moelle osseuse pour la regeneration et la reparation hepatique
US6730775B1 (en) 1999-03-23 2004-05-04 University Of Southern California Methods for limiting scar and adhesion formation
US7652054B2 (en) 2001-05-31 2010-01-26 Vicore Pharma Ab Tricyclic compounds useful as angiotensin II agonists
EP2455388A1 (fr) 2010-11-23 2012-05-23 LanthioPep B.V. Nouveaux agonistes du récepteur 2 (AT2) de type angiotensine et leurs utilisations
WO2021023698A1 (fr) 2019-08-02 2021-02-11 Lanthiopep B.V Agonistes du récepteur de l'angiotensine 2 (at2) destinés à être utilisés dans le traitement du cancer

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5955430A (en) * 1993-09-24 1999-09-21 University Of Southern California Use of angiotensin II fragments and analogs thereof in tissue repair
US5834432A (en) * 1995-06-06 1998-11-10 The University Of Southern California Use of angiotensin II Type 2 receptor agonists in tissue repair
US6335195B1 (en) * 1997-01-28 2002-01-01 Maret Corporation Method for promoting hematopoietic and mesenchymal cell proliferation and differentiation

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000050048A3 (fr) * 1999-02-26 2001-02-01 Univ Pittsburgh Transplantation de moelle osseuse pour la regeneration et la reparation hepatique
US6730775B1 (en) 1999-03-23 2004-05-04 University Of Southern California Methods for limiting scar and adhesion formation
US7652054B2 (en) 2001-05-31 2010-01-26 Vicore Pharma Ab Tricyclic compounds useful as angiotensin II agonists
US8124638B2 (en) 2001-05-31 2012-02-28 Vicore Pharma Ab Tricyclic compounds useful as angiotensin II agonists
EP2455388A1 (fr) 2010-11-23 2012-05-23 LanthioPep B.V. Nouveaux agonistes du récepteur 2 (AT2) de type angiotensine et leurs utilisations
WO2012070936A1 (fr) 2010-11-23 2012-05-31 Lanthiopep B.V. Nouveaux agonistes du récepteur de l'angiotensine de type 2 (at2) et leurs utilisations
US9290540B2 (en) 2010-11-23 2016-03-22 Lanthio Pep B.V. Angiotensin Type 2 (AT2) receptor agonists and uses thereof
US9707268B2 (en) 2010-11-23 2017-07-18 Lanthiopep B.V. Angiotensin type 2 (AT2) receptor agonists and uses thereof
US10214563B2 (en) 2010-11-23 2019-02-26 Lanthiopep B.V. Angiotensin type 2 (AT2) receptor agonists and uses thereof
WO2021023698A1 (fr) 2019-08-02 2021-02-11 Lanthiopep B.V Agonistes du récepteur de l'angiotensine 2 (at2) destinés à être utilisés dans le traitement du cancer

Also Published As

Publication number Publication date
WO1999039743A3 (fr) 1999-09-30
AU2590399A (en) 1999-08-23

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